FACT caught in the act of manipulating the nucleosome

Abstract

The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone ‘FAcilitates Chromatin Transcription’ (FACT, consisting of subunits SPT16 and SSRP1 1 ) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication, and repair 2 . The mechanism by which FACT causes these opposing outcomes is unknown. Here we report two cryo-EM structures of human FACT in complex with partially assembled ‘sub-nucleosomes’, with supporting biochemical and hydrogen-deuterium exchange (HDX) data. FACT is engaged in extensive interactions with nucleosomal DNA and all histones. The large DNA-binding surface on FACT appears to be protected by the C-terminal domains of both subunits, and this inhibition is released by interaction with H2A-H2B, allowing FACT-H2A-H2B to dock onto a (H3-H4) 2 -DNA complex 3 . SPT16 binds nucleosomal DNA and tethers H2A-H2B through its C-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding, and can assume two conformations, depending on whether a second H2A-H2B dimer is present. Our data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage, by facilitating H2A-H2B dimer removal, stabilizing intermediate ‘subnucleosomal’ states, and promoting nucleosome reassembly. Our findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes.

Non-uniform refinement: Adaptive regularization improves single particle cryo-EM reconstruction

“…Indeed, it has been already been successful in helping to resolve a number of interesting new structures. Examples include a structure of FACT manipulating the nucleasome [19], multiple conformations of an ABC exporter [14], mTORC1 docked on the lysosome [24], the respiratory syncytial virus polymerase complex [9], a GPCR-G protein-β-arrestin megacomplex [20], and MERS-CoV/SARS-CoV with neutralizing antibodies [32]. An updated non-uniform refinement algorithm implementation, as described in this work, will be released in an upcoming version of cryoSPARC (www.cryosparc.com).…”

The replisome guides nucleosome assembly during DNA replication

“…FACT was initially identified as a factor that facilitates transcription via the disassembly of H2A-H2B [52,53]. Recently, it was shown that FACT binds both H2A-H2B and H3-H4 and has both nucleosome assembly activity and disassembly activity [54][55][56]. Moreover, FACT is involved not only in the deposition of newly synthesized H3-H4 but also in the recycling of parental histones during replication [34,[57][58][59].…”

Spatial organization of transcribed eukaryotic genes

“…Research by others suggests that highly expressed sequences, such as ribosomal genes in amplified nucleoli of amphibian oocytes (Miller and Beatty, 1969) or sequences on lateral loops of lampbrush chromosomes (Kaufmann et al, 2012), are depleted of nucleosomes as a result of a high polymerase occupancy. Nucleosome eviction is suggested for highly expressed genes (Kulaeva et al, 2010), whereas a moderate expression is apparently compatible with nucleosome retention Gaykalova et al, 2015;Liu et al, 2020). To estimate the compaction level of Tg TLs we measured the contour length of three Tg gene regions after RNA-FISH and found that the Tg compaction level is ca.…”